Construction and secretion of PZ fused with MetLuc from HEK 293 cells. (A) MetLuc cDNA encodes a 219-amino acid polypeptide containing an amino-terminal signal peptide of 17 amino acid residues for secretion (dark lines left panel). PZ cDNA was fused with MetLuc cDNA with (PZ^MetLuc(+Sig)) or without (PZ^MetLuc) the signal peptide of MetLuc. Expression vectors were transfected to HEK 293 cells with pGL3-control, and the cells were incubated without (□) or with (■) Vit.K for 24 hours. MetLuc activity in the culture medium was measured. The data were normalized to the firefly luciferase activity in cell lysates (relative light unit [RLU] against pGL3-control) and were shown as the mean (± SD) for 3 independent experiments (right panel). The numbers depict the residue numbers of the first and last amino acid residues of regions or domains in PZ (in parentheses) or MetLuc (without parentheses). PP, Gla, and PZ_C stand for the propeptide, Gla domain, and C-terminal region of PZ, respectively. (B) PZ in the medium was collected by barium citrate adsorption and subjected to SDS-PAGE under reducing conditions, followed by Western blot analysis using an anti-PZ antibody. The position of human PZ is indicated by an arrow; molecular weight standards are shown by numbers with kilodaltons (kDa).